Splenic-flexure syndrome

* Context.-Microsatellite instability (MSI) due to defective mismatch repair (MMR) genes has been reported in the majority of colorectal tumors from patients with hereditary nonpolyposis colorectal cancer syndrome and in 10% to 15% of sporadic colorectal cancers. The identification of cancers associated with MSI requires classical molecular testing as the gold standard.

Objective.-The aim of this study was to evaluate the role of immunohistochemistry with antibodies directed against 4 MMR proteins as a screening tool for carcinomas with MSI.

Methods.-In this study, 204 formalin-fixed, paraffinembedded colorectal carcinomas were examined for MMR protein expression (hMLH1, hMSH2, hMSH6, and hPMS2) and analyzed for MSI (MSI-H indicates at least 2 of 6 markers affected). These results were correlated with histopathologic parameters.

Results.-Immunohistochemical analysis revealed that

loss of expression of at least 1 protein was present in 17% of cases. One hundred percent of carcinomas that showed high instability (MSI-H) showed loss of expression of hMLH1, hMSH2, or hMSH6. Loss of expression of 2 proteins was present in 59.4% of MSI-H cases, with only 2 combinations, namely, hMLH1/hPMS2 and hMSH2/ hMSH6. Isolated loss of hMSH6 expression was present in 2 MSI-H cases.

Conclusions.-These findings confirm that examination of MMR protein expression by immunohistochemistry is a simple method to diagnose colorectal cancer with MSI. Our data suggest that the study of hMSH6 may be useful, in addition to hMLH1 and hMSH2. Moreover, immunohistochemistry could represent a screening method with which to direct research on the mutations of MMR genes observed in hereditary nonpolyposis colorectal cancer syndrome.

(Arch Pathol Lab Med. 2003;127:694-700)

Colorectal cancer (CRC) is one of the most common forms of cancer in western countries. In most cases, these cancers are seen sporadically, but 5% to 10% of cases are associated with a primary genetic factor. Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common syndrome. It is secondary to an inherited mutation in one of the DNA mismatch repair (MMR) genes (hMLH1, hMSH2, hMSH6, and hPMS2).1,2

The phenomenon of microsatellite instability (MSI) resuits from inactivation, mutational and/or epigenetic, of MMR genes, mostly hMLHl and hMSH2.(3) It is a characteristic molecular finding in most cases of HNPCC and is also observed in a subgroup (10%-15%) of sporadic CRCs.4 It has been shown that MSI tumors, either hereditary or sporadic, have particular morphologic features5-11 and, most importantly, have an improved survival rate relative to microsatellite-stable (MSS) tumors of similar stage,12,13 and may have distinct sensitivity to the action of certain commonly used chemotherapeutic agents.14

Therefore, it appears that recognition of the microsatellite phenotype is becoming important in the management of patients with CRC. Determination of tumor microsatellite status by polymerase chain reaction has been suggested as the best method to recognize MSI-positive CRC.15 However, the demonstration by immunohistochemistry of loss of expression of MMR proteins has been proposed as an alternative method to recognize these tumors.5,9,11,16-32 In most studies, only hMLH1 and hMSH2 proteins were studied, although rare cases of HNPCC are secondary to mutations in other MMR genes. Moreover, the expression of other MMR proteins has not been studied extensively in sporadic CRC.

The aim of this study was to compare the expression of 4 MMR proteins (hMLH1, hMSH2, hMSH6, and hPMS2) to the MSI status determined by polymerase chain reaction in a series of 204 CRCs.

MATERIALS AND METHODS

Samples

Resection specimens were obtained from 204 patients who underwent surgical resection of CRC at Saint-Antoine Hospital (Paris, France) from 1998 to 1999. Site of tumors was determined as rectum, left colon (distal to the splenic flexure), or right colon. Family cancer history was obtained from the clinical charts and was used to identify those families that met either the Amsterdam criteria or extended Bethesda criteria for HNPCC.33,34

Histopathologic Review of Tumors

For all tumors, all hematoxylin-phloxine-safranin-stained slides were reviewed independently by 2 observers (V.R. and J.F.F.). The following variables were assessed: tumor grade (well, moderately, or poorly differentiated), extent of mucin production (mucinous cancers were those containing more than 50% of extracellular mucin), tumor growth pattern (expanding or infiltrating),35 presence of a Crohn-like inflammatory infiltrate (defined as intense lymphoid reaction with numerous lymphoid aggregates with frequent germinal centers),36 and intraepithelial lymphocytes (classified as conspicuous when more than 30 were present per 10 high-power fields).9,37 The tumors were classified in the TNM, Dukes, Astler-Coller, and Jass38 staging systems. A specific medullary type of cancer was recorded, characterized by uniform, poorly differentiated tumor cells arranged in solid sheets with a prominent intratumoral lymphocytic infiltrate.39 Presence of residual adenomatous tissue around the cancer and of distant colonic polyps (hyperplastic, adenomatous, or serrated) was noted.

Immunohistochemical Analysis

One block of formalin-fixed, paraffin-embedded adenocarcinoma tissue was selected in each case. In all cases, this block comprised an area of normal colon mucosa adjacent to the tumor. Four-micrometer sections were affixed to Superfrost Plus slides (CML, Nemours, France) and dried overnight at 37 deg C. After dewaxing and rehydration in distilled water, endogenous peroxidase activity was quenched by incubating the slides in 3% hydrogen peroxide in methanol for (2 x 15 minutes). Sections were subject to heat antigen retrieval by autoclaving in citrate buffer (pH 6.0) for 15 minutes at 750 W and 15 minutes at 150 W. Details of primary antibodies for MMR proteins hMLH1, hMSH2, hMSH6, and hPMS2 are summarized in Table 1. All incubations were performed at room temperature. The following methods were used: avidin-biotin complex method for hMLH1, indirect immunoperoxidase for hMSH2 and hMSH6, and supersensitive detection kit method (BioGenex, San Ramon, Calif) for hPMS2. Immunohistochemical reactions were performed with the Optimax Plus system (BioGenex). Color was developed with aminoethyl-carbazole (Vector, Burlingame, Calif) as the chromogen. The sections were washed in running tap water and lightly counterstained in Mayer hematoxylin. The results were scored without knowledge of the MSI results. Loss of expression was recorded when nuclear staining was absent in all malignant cells, but preserved in normal epithelial and stromal cells. Two observers (V.R. and J.F.F.) assessed all cases independently. The few cases with discrepant scoring were reevaluated jointly on a second occasion, and agreement was reached in all cases.

Microsatellite Instability Testing

The same paraffin block was used for DNA extraction as for immunohistochemistry. DNA was extracted from both normal and malignant tissue embedded in paraffin. Selected areas of malignant tissue comprised more than 70% of malignant cells in all cases. Five to six 20-(mu)m sections were microdissected and placed into microcentrifuge tubes. DNA was extracted with the Qiamp tissue kit (Qiagen, Santa Clarita, Calif). The fluorescent multiplex microsatellite assay was used as previously described with a panel of the following 6 markers: D2S123, D5S346, D17S250, TGFbRII, BAT25, and BAT26.(15) Tumors were considered MSI-H (high) if at least 2 markers showed instability and MSI-L (low) if only 1 marker was unstable.

Statistical Analysis

Qualitative data were compared with the chi^sup 2^ test using Yates correction when appropriate. Quantitative data were expressed as the mean + SEM and compared by the Mann-Whitney test. A P value less than .05 was considered to indicate statistical significance.

RESULTS

COMMENT

In this study, we compared tumor MSI testing by polymerase chain reaction versus tumor immunohistochemistry for assessing the competence of the MMR mechanism of CRCs. In our study, MSI-H tumors were seen in 15.2% of the cases. This rate is slightly higher than percentages previously reported in most studies.9,17,19 This high rate could be partially explained by the presence of 9 patients belonging to an HNPCC family in our population, as in most studies results were considered separately for HNPCC and sporadic MSI-positive CRCs. In our series, 11% of cases had an MSI-L phenotype. As in all reported series, MSI-L CRCs showed no loss of expression of MMR proteins.7,16,17,32 Although some authors have proposed that the MSI-L phenotype reflects a distinctive pathway of development of CRC,40 this theory is very much debated in the literature.41 We do not furnish arguments in favor of this hypothesis, as MSI-L cases were similar to MSS cases in regard to all clinicopathologic characteristics of the tumors.

Our immunohistochemical analysis of MMR enzymes provided a highly specific and sensitive approach to the detection of MSI-H tumors. The performance of immunohistochemistry in comparison with molecular MSI testing is shown in Table 3, which presents the sensitivities, specificities, positive predictive values, and negative predictive values reported in previously published series of CRC and in the present series. As in most other series, we observed a predominance of loss of hMLHl protein in MSI-H tumors, probably due to hypermethylation of the promoter of this gene in sporadic MSI-H cancers.42 In most published studies, only 2 antibodies were used, directed against hMLH1 and hMSH2 proteins (Table 3). These 2 antibodies allowed a diagnosis of MSI with a high specificity (95/-100%) in almost all series, and usually with a high sensitivity, although in some studies this sensitivity was between 70% and 80%, with a relatively high number of false negatives.9,19,21,26 When considering the results of 16 series representing 3494 cases as a whole, the following performances of immunohistochemistry were obtained: sensitivity, 92.4%; specificity, 99.6%; positive predictive value, 98.5%; and negative predictive value, 97.8% (Table 3). Even if molecular MSI testing remains the current gold standard for assessing tumor DNA mismatch pair competency, all these studies confirm that hMLH1 and hMSH2 immunohistochemistry is a sensitive, rapid, and cost-effective method.

Previous studies suggested that one explanation for false-negative cases obtained by immunohistochemistry might be the presence of mutations in still unidentified genes or in other MMR genes.7,17 It seems therefore of interest to study the expression of other MMR proteins, such as hMSH6 and hPMS2, that have been shown to be responsible for rare cases of HNPCC syndrome.

We found 9 cases that did not express hMSH6 protein, of which 7 cases also showed loss of hMSH2 expression and represented the entire group of tumors lacking hMSH2 expression. This association between hMSH2 and hMSH6 expression has already been reported by others.11,32,43 We observed 2 cases with isolated loss of expression of hMSH6 protein, both of which were MSI-H. One patient had an HNPCC syndrome with a germline mutation of the hMSH6 gene, and the second patient was 50 years old. Although we did not have a family history in the latter case, this result might suggest that this patient also had an HNPCC syndrome, as hMSH6 is only rarely implicated in the development of MSI in sporadic CRCs.32 In our series, these 2 cases lacking hMSH6 expression represented 6.5% of MSI-H tumors. Therefore, it seems interesting to study the expression of hMSH6 by immunohistochemistry when a clinical or morphological suspicion of MSI exists, especially when an HNPCC syndrome is suspected, and when there is no loss of either hMLHl or hMSH2 expression, although the MSI status of hMSH6related tumors is still a matter of debate.30,32,44

We also observed a close association between hMLH1 and hPMS2 expression, as 15 of 22 cases with loss of hMLHl also lacked hPMS2, and 15 of 19 cases with loss of hPMS2 also lacked hMLH1. This relationship between hMLH1 and hPMS2 has been reported previously and is considered as secondary to the degradation of hPMS2 in the absence of its binding partner, hMLH1.11,32 We observed 4 cases with isolated loss of hPMS2, but these 4 tumors were all MSS. It appears, therefore, that there is no need to include hPMS2 in the panel of antibodies to be used when looking for MSI by immunohistochemistry.

Although our unselected series mostly included sporadic CRCs, some cases developed in patients with an HNPCC syndrome. In our study, all cases identified by clinical criteria (Amsterdam or Bethesda) showed loss of expression of at least 1 MMR protein. However, it has been demonstrated recently that when a larger number of cases are studied, there is a significant proportion of cancer in HNPCC syndrome with germ-line mutation of hMLHl or hMSH2 that remains positive on immunohistochemistry for these 2 proteins.45 These apparent discrepancies between molecular and immunohistochemical results may be due to the synthesis of mutant proteins that are detected by immunohistochemistry, but that are not functionally active. If these results are confirmed in other series, they may indicate that immunohistochemistry is unlikely to be a primary test for detecting the presence of germline mutations in HNPCC cases. However, as immunohistochemistry is a simple and cheap technique, it can be used to indicate which gene has to be studied for mutation, in case the corresponding protein is not expressed in the tumor.46 In sporadic cancers, false-negative immunohistochemical results are unlikely to occur, because in most MSI-H tumors the promoter for the MMR gene is turned off by hypermethylation, and then no protein is produced. Immunohistochemistry could therefore be a useful screening method to detect MSI-H cases, which may need different chemotherapy.14

In our series, we observed the same clinical and morphologic characteristics of MSI-H CRCs that have been described previously: frequent loss of hMLH1 expression in elderly women, excess of mucinous and medullary types, poorly differentiated cancers, and circumscribed borders.6,9,11 On the contrary, none of these features was related to the MSI-L phenotype. However, although some features are relatively specific for the MSI-H phenotype, they cannot be used as the only detection test, owing to their low sensitivity.6,10 Moreover, we did not confirm the relationship between the MSI-H phenotype and other features previously described in the literature, such as a Crohn-like lymphoid response and an increased number of intraepithelial lymphocytes.6,9-11,37 These discrepancies may be at least partially due to the relatively subjective diagnosis of these features, with criteria variable in different studies. For the intraepithelial lymphocyte count, it may also be explained by the count that we performed on hematoxylin-eosin-stained sections, contrary to most studies with a count made by immunohistochemistry for T lymphocytes.10,37 It seems therefore necessary to include an immunohistochemical study of MMR proteins when the MSI phenotype has to be determined in a large series of patients.

In conclusion, our findings confirm that examination of MMR protein expression by immunohistochemistry is a simple method for diagnosing CRC with MSI. Our data suggest that the study of hMSH6 may be useful, in addition to hMLH1 and hMSH2. Moreover, immunohistochemistry could represent a screening method to direct the research of mutation of MMR genes observed in HNPCC syndrome, and to detect MSI-H sporadic CRCs that may need different chemotherapy.

The authors acknowledge the Fonds de Recherche de la Societe Nationale Francaise de Gastroenterologie (Tours, France), the Fonds de Recherche de la Societe Franqaise de Pathologie (Paris, France), and the Association Charles Debray (Chevigny SaintSauveur, France). Dr Rigau was supported by the Association pour la Recherche sur le Cancer (Villejuif, France).

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Valerie Rigau, MD; Nicole Sebbagh, BS; Sylviane Olschwang, MD, PhD; Francois Paraf, MD, PhD; Najat Mourra, MD; Yann Parc, MD; Jean-Francois Flejou, MD, PhD

Accepted for publication January 9, 2003.

From the Departments of Pathology (Drs Rigau, Mourra, and Flejou, and Ms Sebbagh), Surgery (Drs Olschwang and Parc), AP-HP, Hopital Saint-Antoine, Paris, France; Centre d'Etude du Polymorphisme Humain (CEPH) and Institut National de la Sante et de la Recherche Medicale (INSERM) U434, Paris, France (Drs Rigau, Olschwang, Parc, and Flejou, and Ms Sebbagh); and the Department of Pathology, Hopital Dupuytren, Limoges, France (Dr Paraf).

Reprints: Jean-Fran:ois Flejou, MD, PhD, Service d'Anatomie Pathologique, Hopital Saint-Antoine, AP-HP 184, rue du Faubourg SaintAntoine, 75571, Paris cedex 12, France (e-mail: jean-francois.flejou@ sat.ap-hop-paris.fr).

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